High dose methtrexate with leucovorin rescue is currently being used in a number of clinical protocols for the treatmentof metastatic osteogenic sacroma and a variety of other neoplasms. In both L1210 leukemia and sarcoma 180, Sirotnick and coworkers have clearly demonstrated that high dose methotrexate and leucovorin rescue can be therapeutically optimized by delaying leucovorin administration and minimizing leucovorin doses. The schemes for leucovorin rescue presently being used clinically have been developed on an empiric basis. A thorough knowledge of the pharmacology and pharmacokinetics of calcium leucovorin would enable the clinicians to develop a more rational approach to limiting the doses of llucovorin and improving the therapeutic index of high dose methotrexate/leucovorin rescue therapy. The aim of the present proposal is to develop an accurate, sensitive and reproducible assay for leucovorin in biological fluids. This assay, in order to be clinically useful, will separate leucovorin from both other naturally occurring folates and from the folate antagonists, i.e. methotrexate from both other naturally occurring folates and from the folate antagonists,i.e. methotrexate and aminopterin. Having developed such an assay, we intend to study the absorption, distribution, excretion and pharmacokinetics of leucovorin in humans. In particular, emphasis will be placed on studying the plasma protein binding of leucovorin and drug-drug interactions between leucovorin and methotrexate, antibiotics, antiemetics, and other anti-neoplastic agents. The results of this study will be used to develop a more rational approach towards the use of calcium leucovorin in an effort to minimize leucovorin doses uded and to optimize the therapeutic efficacy of high dose methotrexate with leucovorin rescue therapy.